Alternative splicing coupled to nonsense-mediated decay coordinates downregulation of non-neuronal genes in developing mouse neurons

Background The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. Results We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Conclusions Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-024-03305-8.

Longitudinal RNA-seq data for control-treated TRE-Ngn2 cells were analyzed by DP_GP_cluster [55] and ranked from monotonic downregulation (rank 1) to monotonic upregulation (rank 28) based on the cluster-specific Kendall's trend τ values (see also Fig. S7A).Solid gray lines, temporal dynamics of individual genes in each cluster.Dashed black lines, gene cluster trajectories.S5 for further details.(A) The expression dynamics of Ptbp1 and PTBP1-cotrolled targets in differentiating TRE-Ngn2 cells.The targets shown include neurodevelopmentally upregulated genes with previously characterized NR-CEs (Ptbp2, Dlg4, and Gabbr1) and neurodevelopmentally downregulated genes with NS-CEs shortlisted in this study (Fmnl3, Iqgap1, and Ripk1) (Additional file 5: Table S4).

Fig. S3 :Fig. S4 :
Fig. S3: factR2 analysis of AS-NMD candidates.(A) Transcript biotypes for AS-NMD candidates identified by reanalyzing the GENCODE transcriptome using factR2.(B) The key types of alternatively spliced events examined by factR2.(C) The opposite effects of NMD-stimulating (NS) and NMD-repressing (NR) events on gene expression illustrated for cassette exons, as an example.(D) Distribution of AS-NMD events responding to CHX at both the splicing and gene expression levels at different stages of neuronal differentiation.The custom transcriptome was analyzed as described in Fig. 1E, except using rMATS with |ΔPSI|>0.1 and FDR<0.05cutoffs to shortlist regulated splicing events.

Fig. S5 :
Fig.S5: factR2 events responding to CHX at both the splicing and gene expression levels correlate with developmental changes in gene expression.The two panels show analyses similar to those presented in Fig.2A-B, respectively, but using more stringent Whippet filters to define CHX-responsive factR2 events: |ΔPSI|>0.25 instead of >0.1 and probability>0.95 instead of >0.9.

Fig. S6 :
Fig. S6: Ranking of gene expression trajectories in differentiating TRE-Ngn2 cells.Longitudinal RNA-seq data for control-treated TRE-Ngn2 cells were analyzed by DP_GP_cluster [55] and ranked from monotonic downregulation (rank 1) to monotonic upregulation (rank 28) based on the cluster-specific Kendall's trend τ values (see also Fig.S7A).Solid gray lines, temporal dynamics of individual genes in each cluster.Dashed black lines, gene cluster trajectories.

Fig. S9 :
Fig. S9: Experimental validation of facilitating NS-CEs encoded in genes monotonically downregulated in developing neurons.TRE-Ngn2 cultures were briefly treated with DMSO (odd samples) or CHX (even samples) on differentiation days 0-24 and analyzed by RT-PCR with primers flanking NS-CEs in (A) Ctnnal1, (B) Fbl, (C) Srsf9, and (D) Xpo1 genes.In each panel, the NS-CE containing parts of genes and PhastCons vertebrate conservation tracks are shown on the top; gel analyses of the RT-PCR products are in the middle; and quantifications of NS-CE percent spliced in (PSI) values in DMSO (blue) and CHX (orange) treated samples are at the bottom.Note that in all four cases, NS-CE inclusion tends to increase as a function of development.
(B) One-tailed Fisher's exact test showing that the monotonic downregulation trend (Kendall's τ<-0.75,P<0.05) is overrepresented among genes with PTBP1-controlled facilitating NS-CEs compared to genes containing only non-responsive factR2 events.(C) Conversely, monotonic upregulation (Kendall's τ>0.75, P<0.05) is not enriched among genes with different types of PTBP1-controlled AS-NMD events.(D-F) Count-per-million (CPM) normalized RNA-seq coverage plots for (D) Fnml3, (E) Iqgap1, and (F) Ripk1.The NS-CEs identified by factR2 are highlighted in red.All three genes are strongly downregulated in control-treated differentiating TRE-Ngn2 samples, and this effect is partially rescued by CHX.

Fig. S12 :Fig. S13 :
Fig. S12: PTBP1 downregulation dampens the expression of Fmnl3, Iqgap1 and Ripk1 in an NMD-dependent manner.Mouse ESCs were treated for 48 hours with siRNAs indicated at the bottom of each panel and analyzed by RT-qPCR with primers against constitutively spliced parts of (A) Upf1, (B) Fmnl3, (C) Iqgap1, and (D) Ripk1.Note that PTBP1 knockdown dampens the expression of its target genes and that the knockdown of the key NMD factor UPF1 partially rescues this effect.

Fig. S15 :
Fig. S15: RT-PCR analysis of wild-type and mutant TRE-Ngn2 cells.Wild-type (WT) TRE-Ngn2 cells and the NS-CE mutants introduced in Fig. S14 were treated with DMSO or CHX on differentiation days 0 (ESCs) or 6 (young neurons) and analyzed by RT-PCR with (A) Fmnl3-, (B) Iqgap1-or (C) Ripk1-specific primers designed against the NS-CE-flanking exons.In the wild type, NS-CE-containing mRNAs are readily detectable on day 6 in DMSO-treated samples, becoming a major splice form in the presence of CHX.The ability to produce NMD-sensitive mRNA isoforms is either completely lost (clones D47, B2, A38 and B41) or drastically impaired (clones A15 and E2) in the mutant cells.Asterisks, unspecific amplification products and low-abundance NMD-sensitive transcripts detectable in clones A15 and E2.

Fig. S17 :
Fig. S17: NS-CEs provide a widespread mechanism potentiating gene downregulation in developing neurons.(A-B) High-quality NS-CE events identified by factR2 analysis of developing dentate gyrus granule cells [68] are enriched in developmentally downregulated (A) but not upregulated (B) genes compared to CE-containing genes not predicted by factR2 to undergo AS-NMD.(C-D) The granule-cell NS-CEs encoded in developmentally downregulated, but not upregulated genes tend to be conserved [96] significantly stronger compared to the corresponding groups of CEs not predicted by factR2 to be involved in AS-NMD.